1124
Biochemistry
R. W. KELLERMEYER AND H. G. WOOD
Methylmalonyl Isomerase : A Study of the Mechanism of Isomerization ROBERTW. KELLERMEYERAND ” DG.WOOD From the Department of Biochemistry, Western Reaerve University school of Medicine,Cleveland, Ohio Received July 6,1962
Methylmalonyl-CoA is isomerized to succinyl-CoA by a Blzcoenzymedependent methylmalonyl isomerase and the rearrangement has been shown to occur with a shift of the CoA-carboxyl unit. However, it has not been determined whether the shift of the CoA moiety occurs by an intmmolecular rearrangement, i.e. within the same molecule, or by an intermolecular mechanism. The data from this study show that the rearrangement occurs by an intramolecular shift of the CoA-carboxyl group. Intramolecularly doubly labeled methylmalonyl-CoA was synthesized with C13 in the CoA-carboxyl and the methyl carbons. Eiual amounts of the labeled methylmdonyl-CoA and unlabeled methylmalonyl-CoA were combined and then converted to succinyl-CoA by methylmalonyl isomerase; the succinyl-CoA was then converted to butadiene for mass analysis. The mass analysis showed there was no change in the mass pattern from that of the methylmalonyl-CoA precursor, a finding which could only occur if the CoA-carboxyl group shifted intmmolecularly.
The cobamide (B12-coenzyme)dependent isomerization of methylmalonyl-CoA to succinylCoA is an important reaction in the metabolism ’‘ tissue and bacteria of propionate in (Flavin et al., 1955; Swick and Wood, 1960; Stadtman et al., 1960; Stjernholm and Wood, 1961). It has been demonstrated by C14 labeling experiments (Eggerer et d.,1960; Swick, 1962; Hegre et d.,1962) that the isomerization occurs by a shift of the CoA-carboxyl unit to the methyl p u p (reaction 1). However, these studies do not show whether the rearrangement occurs by an intramolecular shift or an intermolecular shift
C *Hz COOH-LH--COSCoA COOH-CH~-C*H~--COSCOA
of the CoA-carboxyl group. Eggerbr et al. (1960) have postulated that the role of the ‘‘BIZcoenzyme” is to create a free radical of methylmalonyl-CoA through oxidation-reduction of the cobamide cobalt; the CoA-carboxyl then shifts according to the mechanism proposed by Urry and Karasch (1944), presumably by an intramolecular rearrangement (Figure la). Barker and co-workers (Barker et al., 1958; Weisbach
yoo-
700HF-CHB Y=O SCoA
(1)
rCWt++\
HzC-YHz ?=O SCoA
I
c=o I
c= 0 I
SCoA
SCoA
Figure l a
CH3
-
CH3
CoAS,
C,
COaH
Ck
CHZ ,CHIn ,CHzI
HOzC 0
/p
C 0” ‘SCoA
Figure l b FIG. 1.-Examples of intra and intermolecular mechanisms of methylmalonyl-CoA isomerization. Figure la depicts the free radical mechanism postulated by Eggerer et d . (1960)as an example of one type of intramolecular shift of the CoA-carboxyl within the molecule. Figure I b is an example of one type of intermolecular shift of CoA-carboxyl groups (Hegre el ul., 1962).
Vol. 1, No. 6, November, 1962
1125
METHYLMALONYL ISOMERASE
TABLE I SAMPLE CALCULATION OF A PORTION OF THE THEORETICAL MASS SPECTRUM FOR DOUBLY LABELED C L 3BUTADIENE
Butadiene
Propylene
Mass
Proportion of Each Mass in a Single Unit
Butadiene
I
--
-Propylene1
2
3
4
Proportion of Each Mass in a Single Unit
Mass
0.276
54
NO CIS 0.989 X 0.989 X 0.422 X 0.688
42
0.413
1 C'3 0.989 X 0.989 X 0.422 X 0.011 X 0.989 x 0.422 X 0.989 X 0.011 X 0.422 x 0.989 X 0.989 X 0.578 X
0.332 0.668 0.668 0.668
0.574
43
0.521
55 .
2 C'3 0.011 X 0.989 X 0 989 X 0.011 X 0.989 X 0.989 X 0.011 X 0.011 X 0.011 X 0.989 X 0.989 X 0.011 X
44
0.199
0.013 3
45
0.422 X 0.332 0.422 X 0.332 0.578 X 0.332 0.422 X 0.668 0.578 X 0.668 0.578 X 0.668
0.7
sum
x
0.011 X 0.011 X 0.011 X 0.989 X 0.989 X 0.011 X 0.011 X 0.011 X
56
c 1 3
X 0.332 X 0.332 X 0.332 X 0.668
0.422 0.578 0.578 0.578
0.42
10-4
x
10
1,000
1.000 ~
-2
57
-
~
Saying that a unit of butadiene is doubly labeled or singly labzled does not imply that two positions or one position, respectively, of the four carbons will be consistently labeled with C1*. Because there is C13in normal carbon (1.1atoms %) and because the C13 enriched sources such as CO1 are not 100% CI3, it is then apparent that the progressive addition of normal or enriched sources of a one-carbon compound to form a two, three or four carbon compound will result in a spectrum of different combinations or mass values. For example the chance combination of normal carbon atoms (1.1% CI3 and 99.9% CL2)to form a two-carbon compound will result in four different types (four possible combinations) of molecules: C13-C13,C13-C12, C 1 W I 3 ,and C1z-C12, the most prominent type being CLZ-C1*.If a source of carbon atoms enriched with C13 (57.847, CL3,42.2% CIZ) is now added to each of the two-carbon species to create a three-carbon unit (arbitrarily referred to as propylene in this example), there will be eight possible types of three-carbon units; the proportion of each type with respect to mass value is obtained by grouping the different possibilities according to mass, successively multiplying the per cent Cl* or Cl3 of each position within each possibility, and then summing to give the proportion of each mass in a single unit (see boldface type in above table). This process is then continued until the proper number of carbon atoms have been added to make the desired compound. A sample calculation of the different types and proportions of butadiene which would result from a synthesis involving the addition of a CI compound containing 33.2 atoms yo CI3 to a singly labeled propylene with 57.8 atoms yo C is also presented in this table. During the CI addition to these 8 types of propylene, the C13atoms and the C1z atoms would react with each type of molecule, resulting in twice as many types of butadiene (Z4) as propylene. The mass ratios for the butadiene in this example are the theoretical values for butadiene, which is doubly labeled with 57.8 and 33.2 atoms yo C'3. The proportion of masses 54-58 in unlabeled butadiene can be calculated by substituting 0.989 for 0.668 and 0 442 as well as 0.011 for 0.332 and 0 578. The mass patterns for the two species of labeled butadiene that would be encountered in the proposed intermolecular reaction can be obtained by substituting (1) 0.828 for 0.668 and 0.172 for 0.332 for the doubly labeled species 1.1 1.1 57 8 17 2 (C=C---C=C) and (2) 0.989 for 0.422, 0.011 for 0.578, 0.828 for 0.668 and 0.172 for 0.332 1.1 1.1 1.1 17.2 (C=C--C=C). The results of these calculations are given in Table 111.
1126
Biochemistry
R. W. KELLERMEYER AND H. G. WOOD
et al., 1960) have studied a n analogous cobamide dependent reaction in which methy 1 aspartic acid is isomerized to glutamic acid (reaction 2 ) ) but again the nature of the molecular mechanism is unknown.
CHI NHz COOH-A---CH--COOH I
z NH,
COOH-CH~-CH~-C~-COOH
(2)
As defined here a n intermolecular reaction would be said t o occur if there were a complete separation of the CoA-carboxyl from the residual propionic acid moiety and if there were random recombination of the CoA-carboxyl and the threecarbon fragment. Hegre et al. have suggested that this type of intermolecular CoA-carboxyl transfer might occur by a concerted mechanism as shown in Figure l b . By the use of CI3 and mass analysis similar to that used by Wood (1952) and Pomerantz (1958), it is possible to demonstrate whether the molecular rearrangement occurs by an intramolecular (Fig. l a ) or an intermolecular mechanism (Fig. lb). Theoretical Approach to Problem.-Intramolecularly doubly labeled methylmalonyl-CoA was synthesized with Cl3 in both the CoA-carboxyl and the methyl positions. Equal quantities cf labeled and unlabeled methylmalonyl-CoA were then mixed and converted to succinyl-CoA by methylmalonyl isomerase, a conversion whose equilibrium is 10 to 1 in favor of succinyl-CoA (Stjernholm and Wood, 1961). After termination of the reaction, the succinyl-CoA was isolated as succinic acid; the succinic acid was then converted to butadiene in order to remove the oxygen with its various isotopes and tc obtain a gas which is convenient for mass analysis. The feasibility of this type of experiment is dependent on the differentiation of two maas patterns, one for an intramolecular reaction and another for an intermolecular reaction. The type of butadiene mass patterns for each of the proposed mechanisms using intramolecularly doubly labeled (C13) methylmalonyl-CoA can be calculated, thus enabling one to predict if the labeled compounds utilized will be adequate for the experimental design. An example of such a calculation for a butadiene containing 57.8 and 33.2 atoms % C13 in the two labeled positions and the normal complement of CI3 (1.1atoms %) in each of the other two carbons is represented in Table I. The proportions of butadiene with different possible masses are shown on the right side of Table I. These are the theoretical values for a unit of butadiene formed by a doubly labeling process with 57.8 and 33.2 atoms % respectively. Since the theoretical basis for this experiment is dependent on whether the three-carbon propionyl moiety separates from the CoA-carboxyl group, the sample calculation in Table I has been developed to show how the mass proportions of
the three-carbon unit can be derived; for purposes of discussion this three-carbon unit may be considered as propylene. If the CoA-carboxyl group is never separated from the propionyl unit of methylmalonyl-CoA during the isomerization (intramolecular) then pattern of butadiene derived from an the lll~~88 equal mixture of unlabeled methylmalonyl-CoA and methylmalonyl-CoA prepared by a doubly labeling procesa (33.2 atoms % C13 in the CoAcarboxyl and 57.8 atoms % ’ C13 in one position of the propionyl group) would be equal t o the average of the respective mass proportions for each species (Table 111). The calculated mass proportions for the doubly labeled and unlabeled species of butadiene are given in Table 11. If the methylmalonyl-CoA were converted t o succinyl-CoA by an intermolecular reaction as depicted in Figure l b , the specific activity of the C13 in the CoA-carboxyl group of the doubly labeled molecules would be diminished by 50 % . I n effect this would decrease the absolute number of doubly labeled molecules and increase the number of singly labeled molecules, thus creating a mixture of succinyl-CoA (as butadiene) which has an entirely different but predictable mass spectrum from that found for an intramolecular reaction. This mixture of butadiene would be comparable t o equal quantities of (1)a doubly labeled butadiene species with 57.8 atoms % C13in one position of the propylene unit and 17.2 atoms % C13 (1.1 33.2 2 = 17.2) in the carbon corresponding to the CoA-carboxyl group, and (2) a singly labeled species with 17.2 atoms % C13in the carbon corresponding to the CoA-group. The mass proportion for each of these two species was calculated according to the example in Table I and the results are given in Table 11. The predicted mass spectrum for this type of mechanism would be equal to the average of the respective mass proportions obtained when equal amounts of each of the latter two species are mixed (Table 111). It is seen that the values calculated for an intermolecular mechanism differs from those for the intramolecular mechanism. Therefore, the two types of mechanisms can be differentiated by mass analysis using labeled compounds comparable t o those discussed in these sample calculations.
+
EXPERIMENTAL PROCEDURES Synthesis of Doubly Labeled MethylmalonylCoA.-Doubly labeled 2-methylmalonyl-CoA was prepared by the following reactions: KCN NaOH C*HJ -+ C*Ha-C=N --+ 1
2
CsHSCOCl
---C*HpCOONa
LiAIHr 4
+ C*HpCOCI
~
3 HI
KC *N C*HpCHzOH ----f C*H&HpI -+ 5
6
Vol. 1, No. 6,Nouember, 1962
METHYLMALONYL ISOMERASE
1127
TABLE I1 A SUMMARY OF THE PROPORTIONS OF MASSES 54-58 FOR EACHOF THE SPECIES OF BUTADIENE WHICHMIGHT BE ENCOUNTERED I N EITHER A N InterMOLECULAR OR AN I n t r a M O L E C U L A R REACTION Proportion Mass
54
55
56
57
0.957
0 042
0.0007
0.276
0.521
0.191
0.801
0.193
0.342
0.547
Unlabeled butadiene 1.1
1.1
1.1"
1.1
c-c-c-c
5
x
10-b
x
10 - 8
1.0
0.2 X
lo-'
1.0
1
Doubly labeled butadiene 1.1 1.1 5 7 . 8 3 3 . 2
c=c-c=c
Singly labeled butadiene 1 . 1 1 . 1 1 . 1 17.2
c=c-
c-c
0.6 X
lo-?
4.2 X * 1 0 - ~
0.8 X
Doubly labeled butadiene 1.1 1 . 1 5 7 . 8
17.2
c=c-c=c
0,109
0.2
x
Sum
58
lo-'
0.2 X
10-2
0.1
x
1.0 10-4
1.0
Numbers over the carbon atoms denote atoms Cb C13a t that position. These data are obtained according to the sample calculation in Table I: the values for doubly labeled butadiene (57.8 atoms yo C13 and 33.2 atoms 'c C13)were taken directly from Table I.
C*H~CH~-C*EN
NaOH ~
7
-,C*H&H.-C*OONa
ATP, M g - +
-+ C *H,,-CH.-C
propionyl kinase
*OPO.,H,
8
CoASH +
phosphotransace t ylase
C*H,-CHr-C
*OSCoA
9
propionyl carboxylase where * = positions enriched with C I , $ . Methyl iodide (30 mmoles) containing 56.7 atoms % excess C13 was reacted with potassium cyanide and converted to sodium acetate (Little and Bloch, 1950; Sakami, 1955). One-half of the sodium acetate (12 mmoles) was fused in a stream of dry nitrogen in the presence of 30 ml benzoyl chloride; the sodium acetate was converted to acetyl chloride and distilled into a trap cooled by cellosolve and solid CO, (Sakami, 1955). The acetyl chloride was mixed with 10 ml cold redistilled dry diethyl carbitol. An excess of lithium aluminium hydride (0.6 g) was dissolved in 18 ml of diethyl carbitol and added slowly to the acetyl chloride a t 4'. The reaction flask was allowed to come to room temperature and redistilled n-butyl carbitol was added. The flask was connected to an air-cooled distillation column which in turn was connected to a traD cooled in solid COZ and cellosolve. The system was intermittently flushed with nitrogen and heated to boiling several times. The trap containing the ethanol was placed into another train; the alcohol was volatilized and curied with nitrogen gas through boiling 50% HI. H I fumes were removed by a bead tower containing 20% CdC1, and 20% BaC12
and the ethyl iodide was collected in a dry ice trap. An aliquot of the ethyl iodide was converted to ethanol by refluxing with Ag(0H) suspension; the alcohol was separated by distillation and oxidized to acetic acid with a chromic acid solution (Van Slyke and Folch, 1940) and purified by steam distillation and chromatography on a Celite column (Swim and Krampitz, 1950). The sodium acetate was degraded and the C'T content in each carbon atom was determined. The methyl position was found to contain 32.2 atoms % excess. The remaining ethyl iodide was reacted with KCN (10% excess) containing 32.2 atoms % excess CI3 (Little and Bloch, 1950; Sakami, 1955). The propionyl nitrile was hydrolyzed and the sodium propionate was purified by steam distillation and Celite column Chromatography (Swim and Krampitz, 1950). The sodium propionate 1,3-C1
